Abstract

BACKGROUND:

p68 (Ddx5) and p72 (Ddx17) are highly related members of the DEAD box family and are established RNA helicases. They have been implicated in growth regulation and have been shown to be involved in both pre-mRNA and pre-rRNA processing. More recently, however, these proteins have been reported to act as transcriptional co-activators for estrogen-receptor alpha (ER alpha). Furthermore these proteins were shown to interact with co-activators p300/CBP and the RNA polymerase II holoenzyme. Taken together these reports suggest a role for p68 and p72 in transcriptional activation.

RESULTS:

In this report we show that p68 and p72 can, in some contexts, act as transcriptional repressors. Targeting of p68 or p72 to constitutive promoters leads to repression of transcription; this repression is promoter-specific. Moreover both p68 and p72 associate with histone deacetylase 1 (HDAC1), a well-established transcriptional repression protein.

CONCLUSIONS:

It is therefore clear that p68 and p72 are important transcriptional regulators, functioning as co-activators and/or co-repressors depending on the context of the promoter and the transcriptional complex in which they exist.

Effect of GAL4-tagged p68 and p72 on transcriptional activity as measured by CAT assays using the TK-CAT, MLP-CAT and SV40-CAT promoter-reporter plasmids, each harbouring 5 copies of a GAL4 binding site fused to the promoter. The pcDNA3-GAL4 expression vector (pcG4) was used as a control. In each case U2OS cells were co-transfected with pcG4 or plasmids expressing GAL4-tagged p68/p72 (p68G4/p72G4) and the appropriate promoter-reporter construct. The amounts of DNA transfected were: pcG4-, p68G4-/p72G4- 1 μg; TK-CAT- 2.5 μg; MLP-CAT- 9 μg; SV40-CAT- 0.5 μg. The amount of DNA used had been previously titrated to achieve appropriate levels of baseline CAT activity. a) Transcriptional repression by p68 and p72 as measured by CAT activity, which is shown as % conversion of 14C-labelled chloramphenicol to acetylated forms. The data represent results from 5 independent assays, which were each performed in triplicate. b) Western blot, using a GAL4-specific antibody, showing expression levels of the pcG4, p68G4 and p72G4 plasmids.

Control CAT assays to examine repression of the TK-CAT promoter-reporter in U2OS cells by p68/p72. The amounts of DNA transfected in each assay are indicated below and in all cases the % conversion of 14C-labelled chloramphenicol to acetylated forms is shown as an average of three independent experiments. a) Effect of untagged p68/p72 on TK-CAT transcription. 7.5 μg of control pcDNA3 vector, pcDNA3-p68 (p68) or pcDNA3-p72 (p72) were co-transfected with 2.5 μg of TK-CAT. b) Effect of GAL4-tagged p68 and p72 on transcriptional activity of a TK-CAT promoter-reporter which incorporated a 1.6 kb DNA 'spacer' between the GAL4 binding sites and the promoter (TK-S-CAT). 1 μg of pcDNA3-GAL4 (pcG4) or GAL4-tagged p68/p72 (p68G4/p72G4) were co-transfected with 5 μg of TK-S-CAT. The amount of TK-S-CAT had previously been titrated to achieve an appropriate baseline level of CAT activity. c) Titre of repression of TK-CAT activity by GAL4-tagged p68/p72. 2.5 μg of TK-CAT were co-transfected with different amounts of pcG4 vector, p68G4 and p72G4 as indicated. d) Effect of p300 and CBP on repression of TK-CAT transcription by GAL4-tagged p68/p72. 2.5 μg of TK-CAT were co-transfected with 1 μg of pcG4 vector, p68G4 or p72G4 together with 6.5 μg of either bluescript (as control) or p300/CBP.

Deletion mapping of potential repression/activation domains in a) p68 and b) p72 as observed in CAT assays using the TK-CAT promoter-reporter plasmid. The pcDNA3-GAL4 expression vector (pcG4) and full-length GAL4-tagged p68/p72 were used as controls. All p68/72 deletion derivatives were expressed as GAL4-tagged fusion proteins in pcG4 and included the amino acids indicated. Additional proteins tested in this assay included the ATPase/helicase GAL4-tagged inactive mutants of p68/p72 (p68N/p72N) and the alternative upstream initiation product of the p72 gene (p82). The amounts of DNA used in the transfections were; TK-CAT- 2.5 μg; pcG4 and all p68/p72 constructs- 1 μg. The % conversion of 14C-labelled chloramphenicol to acetylated forms is shown as an average of five independent experiments. c) Diagram correlating the deletion end-points to the position of the motifs conserved in the DEAD box family of proteins.

The involvement of HDAC activity in transcriptional repression by p68/p72. a) Relief of p68/p72 repression of MLP-CAT transcription by TSA. 1 μg of pcDNA3-GAL4 (pcG4) or GAL4-tagged p68/p72 (p68G4/p72G4) were co-transfected with 9 μg of MLP-CAT and TSA was added 16 hr after transfection, at a final concentration of 300 nM. The values for p68G4 and p72G4 are given relative to the baseline value for the pcG4 vector control, which was set at 1, and represent the average from three experiments. b) Immunoprecipitation/western blotting of myc-tagged p68 and p72 from 293 cells expressing these proteins. Myc-tagged proteins were immunoprecipitated with the anti-myc epitope antibody, 9E10, and western blotted with the same antibody to detect the presence of p68-myc and p72-myc fusion proteins. A myc-tagged pSG5 vector control is included. pSG5, p68 and p72 all refer to myc-tagged versions. H denotes cross reaction with the antibody heavy chain. Molecular weight markers (in kDa) are indicated. Equal amounts of these immunoprecipitated proteins were used in the HDAC activity assay shown in c. c) HDAC activity assay of immunoprecipitated p68 and p72 (see b). HDAC activity in the presence and absence of TSA is shown relative to that of the myc-tagged pSG5 vector control, which was set at 1, and represent the average from three experiments.

Western blots showing gel filtration elution profiles of p68, p72 and HDAC1. p68, p72 and HDAC1 in the fractions were detected by western blotting using appropriate antibodies. Note that the antibody raised against p72 also recognises p82 and cross-reacts with NFAR-2 [15]. All lysates had been treated with DNase and RNase prior to gel filtration. The void volume and elution position of the Pharmacia FPLC size markers are indicated, as are molecular weight markers (in kDa).

Co-immunoprecipitation of a) p68 and b) p72 with HDAC1. a) HDAC1 was immunoprecipitated from U2OS nuclear extracts using an HDAC1-specific antibody. Immunoprecipitated proteins were separated by SDS-PAGE and the presence of HDAC1 and associated p68 was detected by western blotting with p68- and HDAC1-specific antibodies. b) HDAC1 was immunoprecipitated from nuclear extracts of 293 cells expressing myc-tagged p72 using an HDAC1-specific antibody. Immunoprecipitated proteins were separated by SDS-PAGE and the presence of HDAC1 and associated myc-tagged p72 was detected by western blotting with HDAC1- and myc epitope- specific antibodies. In both experiments a control immunoprecipitation (IP) was performed using an irrelevant rabbit IgG. An aliquot of nuclear extract (NE) was also included in the western blots (west.) as an additional control. H denotes cross reaction with the antibody heavy chain. Molecular weight markers (in kDa) are indicated.

a) Reciprocal co-immunoprecipitation of p68 and p72 with HDAC1. Myc-tagged p68 and p72 were immunoprecipitated from nuclear extracts of 293 cells expressing these proteins using a myc epitope-specific antibody and associated HDAC1 was detected by western blotting with an HDAC1-specific antibody. 293 cells, which had not been transfected, were used as control. b) Co-immunopreciptiation of p68/p72 deletion derivatives with HDAC1. HDAC1 was immunoprecipitated from nuclear extracts of 293 cells expressing GAL4-tagged p68 and p72 deletion derivatives, which encompass residues 1–478 and 1–474 of p68 and p72 respectively. Associated p68/p72 were detected by western blotting with a GAL4-specific antibody. 293 cells, which had not been transfected, were used as control. NE-nuclear extract, IP-immunopreciptiation. Molecular weight markers (in kDa) are indicated.